siRNA / miRNA gene silencing Mouse B16 Melanoma cells

DNA microarrays enable researchers to monitor the expression of thousands of genes simultaneously. However, the sensitivity, accuracy, specificity, and reproducibility are major challenges for this technology. Cross-hybridization, combination with splice variants, is a prime source for the discrepancies in differential gene expression calls among various microarray platforms. Removing (either from production or downstream bioinformatic analysis) and/or redesigning the microarray probes prone to cross-hybridization is a reasonable strategy to increase the hybridization specificity and hence, the accuracy of the microarray measurements.

Get tips on using pET-21b(+)-mTNFα to perform Protein Expression Prokaryotic cells - E. coli mouse TNF-(α)

Get tips on using RNeasy Fibrous Tissue Mini Kit to perform RNA isolation / purification Cells - primary mouse ventricles

Get tips on using RNeasy Fibrous Tissue Mini Kit to perform RNA isolation / purification Cells - primary mouse cardiomyocytes

Get tips on using Pierce™ BCA Protein Assay Kit to perform Protein quantification Mammalian cells - Mouse macrophages

Get tips on using Pierce™ BCA Protein Assay Kit to perform Protein quantification Mammalian cells - Mouse microglia

Get tips on using Quant-iT™ RNA Assay Kit to perform RNA quantification Fuorimetric - mouse glial cells

The process of RNA extraction from bacteria, in general, involves an RNA-protective, effective lysis of bacterial cell wall (which may pose difficulties). EDTA promotes loss of outer membrane to provide lysozyme with access to peptidoglycan. Another common method for cell wall lysis is mechanical disruption using a homogenizer (applied for gram-positive bacteria and some strains of gram-negative bacteria). Following lysis, it is necessary to disrupt protein-nucleic acid interactions, which can be achieved by adding sodium dodecyl sulfate (SDS). Next step involves using phenol-chloroform-isoamyl alcohol extraction, where RNA can be obtained from the bottom organic phase, the top phase consists of DNA and the interphase contains proteins. Isoamyl alcohol is an inert and optional addition to this mixture and is added as an anti-foaming reagent to reduce the interphase. Following RNA extraction, the samples should be checked for its quality by gel electrophoresis (23S and 16S rRNAs and 5s rRNA and tRNA bands) or UV spectrophotometric or fluorescence methods.

Get tips on using pGL3-Basic Vector to perform Reporter gene assay luciferase - human embryonic stem cells